The Moss Physcomitrella Patens Is Hyperresistant to DNA Double-Strand Breaks Induced by Γ-Irradiation

G C A T T A C G G C A T genes Article The Moss Physcomitrella patens Is Hyperresistant to DNA Double-Strand Breaks Induced by γ-Irradiation Yuichiro Yokota * and Ayako N. Sakamoto Department of Radiation-Applied Biology Research, Takasaki Advanced Radiation Research Institute (TARRI), National Institutes for Quantum and Radiological Science and Technology (QST), Watanuki-machi 1233, Takasaki, Gunma 370-1292, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-27-346-9113 Received: 31 October 2017; Accepted: 31 January 2018; Published: 7 February 2018 Abstract: The purpose of this study was to investigate whether the moss Physcomitrella patens cells are more resistant to ionizing radiation than animal cells. Protoplasts derived from P. patens protonemata were irradiated with γ-rays of 50–1000 gray (Gy). Clonogenicity of the protoplasts decreased in a γ-ray dose-dependent manner. The dose that decreased clonogenicity by half (LD50) was 277 Gy, which indicated that the moss protoplasts were 200-times more radioresistant than human cells. To investigate the mechanism of radioresistance in P. patens, we irradiated protoplasts on ice and initial double-strand break (DSB) yields were measured using the pulsed-field gel electrophoresis assay. Induced DSBs linearly increased dependent on the γ-ray dose and the DSB yield per Gb DNA per Gy was 2.2. The DSB yield in P. patens was half to one-third of those reported in mammals and yeasts, indicating that DSBs are difficult to induce in P. patens. The DSB yield per cell per LD50 dose in P. patens was 311, which is three- to six-times higher than those in mammals and yeasts, implying that P. patens is hyperresistant to DSBs. Physcomitrella patens is indicated to possess unique mechanisms to inhibit DSB induction and provide resistance to high numbers of DSBs. Keywords: moss; Physcomitrella patens; protoplasts; γ-rays; radiosensitivity; DNA double-strand breaks; pulsed-field gel electrophoresis 1. Introduction Organisms show diverse radiosensitivity in a broad range [1,2] and are thus expected to possess various genome maintenance strategies. Terrestrial plants are often hyperresistant to ionizing radiation, although the cells contain relatively large contents of genomic DNA, which is the most important biological target of radiation. After Arabidopsis thaliana and tobacco (Nicotiana tabacum) dry seeds were irradiated with electron radiation or γ-rays and seedlings grown to measure survival rates, the doses that decreased the survival rate by half (LD50) were about 1250 and 700 gray (Gy), respectively [3,4]. However, few studies have assessed the radiosensitivity of single plant cells and thus it remains unclear whether plants are equipped with hyperresistance to radiation at the cellular level. In a previous study, we investigated the clonogenicity of protoplasts derived from the tobacco BY-2 cell line and determined that tobacco protoplasts were 10-times more resistant to radiation than mammalian cells [5]. Ionizing radiation induces various types of DNA damage [6]. Among the types of damage, double-strand breaks (DSBs) are difficult to repair accurately and the impacts are the most serious biologically. To study the mechanism of induction and/or repair of DSBs, several techniques have been developed to detect DSBs [7]. Pulsed-field gel electrophoresis (PFGE) is one of the quantitative methods developed. We previously performed a comparative analysis of initial DSB yields between tobacco protoplasts and Chinese hamster CHO-K1 cells, and reported that the yield per Gb DNA per Gy (Gb DNA−1 Gy−1) in tobacco was only one-third of that induced in the Chinese hamster [8]. Friesner et al. [9] measured DSBs in A. thaliana using an antibody to phosphorylated H2A histone Genes 2018, 9, 76; doi:10.3390/genes9020076 www.mdpi.com/journal/genes Genes 2018, 9, 76 2 of 11 family, member X (γ-H2AX), a biological marker of DSBs, and reported that the DSB yield in A. thaliana was similar to that in tobacco and less than those in mammals. Furthermore, at least two-times more DSBs are necessary to inactivate tobacco and A. thaliana cells compared with mammalian cells [8,9]. However, the mechanism of plant hyperresistance to DSBs is unknown. The ultimate goal of our research is to elucidate the mechanisms of plant cell hyperresistance to ionizing radiation. The moss Physcomitrella patens is an ideal model terrestrial plant to achieve this objective; a complete genome sequence for P. patens is available [10], protoplasts are readily isolated and cultured [11], and gene targeting is feasible [12]. The aims of the present study were to analyze the relationship between radiosensitivity and the DNA DSB induction rate of P. patens cells and to elucidate the mechanism of hyperresistance to ionizing radiation. Isolated protoplasts were irradiated with γ-rays, the radiosensitivity was evaluated, and the initial DSB yield was quantified. This is the first report that quantifies the initial DSB yield in a moss and compares it with hyperresistance to radiation. 2. Materials and Methods 2.1. Suspension Culture A culture of the moss P. patens was kindly gifted from Daisuke Takezawa (Saitama University, Saitama, Japan). The protonemata were disrupted with a homogenizer (Microtec Co., Ltd., Chiba, Japan) and suspension-cultured in a basal medium developed specifically for the moss (called as BCDAT) without agar [11], on a reciprocal shaker at 120 rpm and 23 ◦C under continuous light. The suspension culture was subcultured every week in a 1:5 dilution. 2.2. Protoplast Isolation The suspension-cultured protonemata were collected by centrifugation at 1000× g for 5 min. Centrifugation was always performed at 20 ◦C in the present study. To isolate protoplasts, the cell wall of the collected protonemata was digested in enzyme solution consisting of 0.5% Macerozyme R-200, 1% Cellulase “Onozuka” RS (Yakult Pharmaceutical Industry, Tokyo, Japan), and 8% mannitol, and incubated for 3 h at 23 ◦C without light. The isolated protoplasts were filtered through a 40-µm-diameter cell strainer (Corning, NY, USA), washed in 8% mannitol solution three times, and collected at 200× g for 5 min. 2.3. Analysis of Cell Cycle Phase Distribution Protoplasts isolated from the protonemata suspension culture were resuspended in nuclei extraction buffer (CyStain UV Precise P, Partec GmbH, Münster, Germany) and incubated for 30 min. Extracted nuclei were filtered through a 30-µm mesh nylon sieve, and stained with four volumes of 40,6-diamidino-2-phenylindole (DAPI) staining buffer (CyStain UV Precise P, Partec) for 30 min. The cell cycle in P. patens protoplasts was analyzed using a flow cytometer (type PA, Partec). As a reference, cell nuclei extracted from rosette leaves of A. thaliana ecotype Columbia were stained and analyzed under the same conditions. 2.4. g-Irradiation The protonemata and protoplasts were irradiated with γ-rays [13]. Irradiation experiments were performed at room temperature in the Cobalt-60 irradiation facility of the Takasaki Advanced Radiation Research Institute (TARRI), National Institutes for Quantum and Radiological Science and Technology (QST), Takasaki, Japan. 2.5. Growth Assay Protonemata were irradiated for 10 min at dose rates of between 10 and 50 Gy/min. Irradiated protonemata were suspension-cultured for one week. The protonemata were collected on Genes 2018, 9, 76 3 of 11 paper disks by filtration. The collected protonemata were air-dried overnight, and then the dry weight was measured using a microbalance. The dry weight at day 0 was subtracted from that at day 7 to estimate the growth potential for one week. The increased weight of the irradiated protonemata was normalized by dividing it by that of sham-irradiated protonemata. 2.6. Colony Formation Assay The colony formation assay is a conventional method used to measure the survival rate of single mammalian cells after treatment with abiotic stresses [14], which we applied for measurement of the survival rate of moss protoplasts. Isolated protoplasts were resuspended in the protoplast liquid medium [11] and incubated for 24 h at 23 ◦C without light. After incubation, the protoplasts were collected by centrifugation at 200× g for 5 min. The collected protoplasts were resuspended in a protoplast regeneration medium (PRM/T) without agar [11], counted using a hemocytometer, and the density was adjusted to 1000–5000 cells/mL. Protoplasts were irradiated for 10 min at dose rates of between 5 and 45 Gy/min. Immediately after irradiation, the protoplasts were mixed with an equal volume of PRM/T medium supplemented with 1.6% SeaPlaque low-melting agarose (Lonza, Basel, Switzerland). One milliliter of the mixture was dropped and spread on 60-mm-square hydrated cellophane (Bio-Rad Laboratories, Hercules, CA, USA), which was laid on PRM/T medium supplemented with 0.8% agar. The protoplasts were cultured at 23 ◦C under light for two to three weeks to allow surviving protoplasts to form visible colonies including 50 or more cells. Three to four days after the spreading, the cellophane and protoplasts were moved onto BCDAT medium supplemented with 0.8% agar. The colony formation rate was calculated by dividing the number of colonies by that of inoculated protoplasts. The survival rate was derived by dividing the colony formation rate of irradiated protoplasts by that of sham-irradiated protoplasts. 2.7. Pulsed-Field Gel Electrophoresis Assay Protoplasts were irradiated immediately after isolation for the DSB assay to avoid, if any, an effect of cell wall regeneration. Isolated protoplasts were resuspended in 8% mannitol, counted using a hemocytometer, and the density was adjusted to 2.5 × 106 cells/mL. The protoplast suspension was mixed with an equal volume of 8% mannitol supplemented with 1.5% agarose GB (Nippon Gene, Tokyo, Japan). The mixture was poured into plug molds (80 µL volume per mold; Bio-Rad Laboratories) and solidified at 4 ◦C.

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